1. Field of the Invention
This invention relates to transgenic plants that are genetically engineered to contain a DNA sequence encoding at least one polypeptide having a 2,5A synthetase activity thereby providing to said plants resistance against multiple taxonomic virus types. Moreover, this invention relates to a process for the production of said transgenic plants and to the use of said genetically engineered DNA sequence.
2. Description of Related Art
Several methods for the construction of virus resistant plants are described in the state of the art. Genetically engineered resistance to a number of plant viruses has been reported by expressing coat protein of a respective plant virus in transgenic plants (Beachy, et al., Annu. Rev. Phytopathol., 28:451-474, 1990).
A substantial virus-resistance to several plant viruses has also been demonstrated by expression of specific viral antisense RNA in transgenic plants (Cuozzo, et al, Bio/Technology, 6:549-557 1988; Hemenway, et al., EMBO J., 7: 1273-1280, 1988). The viral antisense RNA exhibits its function either by hybridizing to specific viral DNA or RNA sequences and thus blocking further reactions which are important for the virus propagation, or by ribozyme activity which results in a specific cleavage of viral RNA upon hybridization to said viral RNA.
The main drawback of the above mentioned methods for the construction of virus-resistant transgenic plants is that said transgenic plants are resistant to only one virus or a specific taxonomic group of viruses.
The 2,5A oligoadenylate pathway is part of the antiviral response system induced by interferons in mammalian cells (Lengyel, Annu. Rev. Biochem., 51:251-282, 1982). The key enzyme of the pathway, the 2,5A synthetase, polymerizes ATP to a family of oligonucleotides, the 2,5A. Virus replication is inhibited due to the degradation of viral RNA by the specific 2,5A-activated ribonuclease, RNase L.
In the mammalian system, the 2,5A synthetase polymerizes ATP in the presence of double-stranded RNA (dsRNA, for example replicative intermediates of RNA viruses) to produce a family of oligonucleotides with the general structure PPP(A2'p5').sub.n A with n.gtoreq.2, abbreviated 2,5A. These oligonucleotides possess 2'-5' phosphodiester bonds that are unusual in comparison with ordinary 3'-5' links in the nucleic acids. Two other enzymes involved in the 2,5A system are: (i) 2'-5' phosphodiesterase, which degrades 2,5A, and (II) 2,5A-dependant ribonuclease (RNase L). The 2,5A synthetase is expressed as a inactive enzyme. For its activation, the presence of dsRNA is required. Only dsRNA molecules having a length of at least about 50 base pairs and with no more than one mismatch per 45 nucleotides can activate the synthesis of 2,5A (M. A. Minks, et al., J. Biol. Chem., 254:10180-10183, 1979). Viral RNA has been shown to be a very potent activator of the 2,5A pathway (P. Lengyel, J. Interferon Res. 7:511-519, 1987). Actually, 2,5A is not a single compound, but a mixture of oligoadenylates with different chain lengths and states of phosphorylation. Oligomers with at least three residues are required to activate RNase L. Another requirement for 2,5A activation of RNase L in mammalian cells is a 5' di- or tri-phosphate group. The existence of nonphosphorylated "core" 2,5A molecules in cells also has been reported, but they neither bind to nor activate the RNase L. Activation of the RNase L in mammalian cell extracts is observed already at nanomolar concentrations of 2,5A (I. M. Kerr, J. Interferon Res., 7:505-510, 1987). Due to the activity of 2'-5'-phosphodiesterase in cells, the activation of the 2,5A-dependent RNase is transient without the persistent de novo synthesis of 2,5A.
At least some components of the 2,5A pathway have been detected in organisms other than mammals such as birds, reptilia and amphibia, insects, yeasts and even bacteria (Stark, et al., Nature, 278:471-473, 1979; Cayley, et al., Biochem. Biophys. Res. Comm., 108:1243-1250, 1982; Laurence, et al., Proc. Natl. Acad. Sci. U.S.A., 81:2322-2326, 1984).
In plants, an antiviral factor (AVF), the production of which is stimulated by virus infection, was partially purified and its gene appeared to be homologous to human .beta.-interferon; Sela, et al., in: Plant resistance to Viruses: Ciba Symposium, No. 133 pp. 109-119, 1987. Treatment of tobacco mosaic virus (TMV)-infected tobacco protoplasts with human interferon led to the inhibition of TMV-replication; Sela, Methods in Enzymology, 119:744-752, 1986. Moreover, it has been demonstrated that 2,5A can inhibit TMV replication in tobacco plants; Devash, et al., J. Biol. Chem., 259:3482-3486, 1984. DNA sequences homologous to human 2,5A synthetase were also found in tobacco genomic DNA; Sela, supra. The absence of (2'-5')pppA.sub.n A-binding proteins in plants has been independently demonstrated (P. J. Cayley, et al., Biochem Biophys. Res. Commun., 108:1243-1250, 1982). Furthermore, it has been reported that interferon produced in transgenic plants does not inhibit virus propagation; De Zoeten, et al., Virology, 172:213-222, 1989.
A 2,5A dependent RNAse, which is part of the 2,5A oligoadenylate pathway in mammals, has not yet been reported in plants in the state of the art. This has led to the conclusion that 2,5A does not inhibit virus infection in plants via a 2,5A dependent endonuclease; Devash, et al., Biochemistry, 24:593-599, 1985. A probe of the human 2-5A synthetase was shown to hybridize to tobacco genomic DNA and mRNA from TMV infected tobacco (I. Sela, et al. in: Evered D., et al. Eds., Plant Resistance to Viruses., J. Wiley, Chichester, pp 109, 119, 1987), although 2,5A synthetase activity had not been detected in tobacco earlier (Cayley, supra). The partially purified ATP-polymerizing plant enzyme also reacted with antibodies to human 2,5A synthetase (Sela, et al., supra). Recently, exogeneous nonphosphorylated 2-5 A molecules longer than trimers were demonstrated to induce both increased cytokinin activity and the synthesis of pathogenesis-related and heat shock proteins in tobacco and wheat (O. N. Kulacva, et al., Plant Mol. Biol., 20:383-393, 1992).
In summary, the prior art does not permit the conclusion that the 2,5A oligoadenylate pathway can be used as a basis for constructing transgenic plants displaying multiple virus resistance. Thus, the technical problem of the present invention is to provide a transgenic plant displaying resistance to multiple virus taxonomic groups using parts of the 2,5A oligoadenylate pathway.
The solution to the above technical problem is achieved by providing the embodiments characterized in the claims.
One object of the present invention relates to a transgenic plant displaying multiple virus resistance which contains a genetically engineered DNA sequence encoding at least one polypeptide having a 2,5A synthetase activity, wherein said polypeptide upon expression is capable of activating an endonuclease causing degradation of viral RNA.